Journal: Biology of reproduction

Article Title: The activin-follistatin system in the neonatal ovine uterus.

doi: 10.1095/biolreprod.103.016287

Figure Lengend Snippet: FIG. 2. Expression of ActRs in the neonatal ovine uterus. In situ hybridization and immunohistochemical analysis of ActRIA (A) and ActRII (B) and immunohistochemical analysis of ActRIB (C) in the uterus. Except for ActRIB, representative photomicrographs of in situ hybridization results are presented in bright-field and dark-field illumination (left). Melanocytes (Mel) in the endometrium appear white in the dark-field images and black in bright-field images, but they do not express subunit mRNA. Representative photomicrographs of immunohistochemical results are presented for the upper and lower portions of the uterine wall (right). As a negative control, mouse IgG (mIgG) was substituted for the primary antibody. M, Myometrium; S, stroma. Bars 5 50 mm.

Article Snippet: Mouse anti-human monoclonal antibody to follistatin (catalog no. MAB669), ActRIA (catalog no. MAB637), ActRIB (catalog no. MAB222), and ActRIIA/B (catalog no. MAB3391) were from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Expressing, In Situ Hybridization, Immunohistochemical staining, Negative Control

Journal: REPRODUCTION

Article Title: Postnatal uterine development in Inverdale ewe lambs

doi: 10.1530/rep-07-0323

Figure Lengend Snippet: Figure 3 Immunohistochemical localization of androgen receptor (AR) and activin receptors (ActRIA, IB and II) in the uteri of CC and II ewes. GE, glandular epithelium; LE, luminal epithelium; S, stroma. The scale bar indicates 100 mm at low magnification and 25 mm at high magnification.

Article Snippet: Rabbit polyclonal antibody to androgen receptor (sc-816, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), and mouse monoclonal Reproduction (2008) 135 357–365 antibodies to ActRIA (MAB637; R&D Systems Inc.), ActRIB (MAB222; R&D Systems), and ActRII (MAB3391; R&D Systems) were used for immunohistochemistry (Carpenter et al. 2003a, Hayashi et al. 2003, Juengel et al. 2006).

Techniques: Immunohistochemical staining

Journal: bioRxiv

Article Title: An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro/adipogenic progenitors in fibrodysplasia ossificans progressiva mice

doi: 10.1101/2021.07.23.451471

Figure Lengend Snippet: ( a ) Mouse myoblast C2C12 cells express Acvr1 and Alk3 but not Alk1 or Alk6 , as detected by RT-qPCR. (b) Surface expression of ACVR1 and ALK3 on C2C12 cells, as detected by flow cytometry. (c) Monoclonal antibody JAB0505 binds to parental C2C12 cells, but not Acvr1 KO cells, as assessed by flow cytometry. (d) JAB0505 inhibits BMP signal activation in wild-type and ACVR1(R206H)-overexpressing C2C12 cells in a dose-dependent manner, as determined by quantification of BRE-luciferase activity.

Article Snippet: A synthetic DNA construct encoding mouse ACVR1 with the R206H mutation (G to A at nucleotide position 617) was assembled from synthetic oligonucleotides and PCR products and cloned into the pCMV6 entry mammalian expression vector (Origene), which also encodes a neomycin resistance marker.

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Activation Assay, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro/adipogenic progenitors in fibrodysplasia ossificans progressiva mice

doi: 10.1101/2021.07.23.451471

Figure Lengend Snippet: ( a ) Representative µCT images of HO in Acvr1 FLEx(R206H)/+ ;CAG-Cre ERT2 mice 20 days after cardiotoxin-induced injury of the gastrocnemius muscle (Untreated, n = 3; JAB0505, n = 4). (b) Representative µCT images and volumetric quantification of HO (pseudocolored green) in Acvr1 tnR206H/+ ;Tie2-Cre mice 21 days after pinch injury of the gastrocnemius muscle (Untreated, n = 11; JAB0505, n = 10. Bars represent ± SD. p = 0.001, unpaired t-test). (c) Alcian blue staining for cartilage (arrows) and immunohistochemical staining of ACVR1 (brown, arrows) on cross-sections of pinch-injured muscle from untreated wild-type mice, untreated Acvr1 tnR206H/+ ;Tie2-Cre mice (lesional region denoted by asterix), and JAB0505-treated Acvr1 tnR206H/+ ;Tie2-Cre mice. Centrally located myofiber nuclei (arrowheads) identify regenerated fibers, which were abundant in wild-type mice, rare in Acvr1 tnR206H/+ ;Tie2-Cre mice, and undetected in JAB0505-treated Acvr1 tnR206H/+ ;Tie2-Cre mice. AB/E, Alcian blue/eosin. Sections processed for immunohistochemistry were counterstained with hematoxylin. (d) Low magnification images of lower hindlimb cross-sections of Acvr1 tnR206H/+ ;Tie2-Cre mice at day 14 post-injury. As compared to HO (asterix) of untreated Acvr1 tnR206H/+ ;Tie2-Cre mice at this stage, boney HO was less prevalent in JAB0505-treated Acvr1 tnR206H/+ ;Tie2-Cre mice, which also exhibited broader distribution of cartilaginous HO (arrows). Sections were stained with Alcian blue and eosin.

Article Snippet: A synthetic DNA construct encoding mouse ACVR1 with the R206H mutation (G to A at nucleotide position 617) was assembled from synthetic oligonucleotides and PCR products and cloned into the pCMV6 entry mammalian expression vector (Origene), which also encodes a neomycin resistance marker.

Techniques: Staining, Immunohistochemical staining, Immunohistochemistry

Journal: bioRxiv

Article Title: An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro/adipogenic progenitors in fibrodysplasia ossificans progressiva mice

doi: 10.1101/2021.07.23.451471

Figure Lengend Snippet: ( a ) Osteogenic differentiation of monolayer R206H-FAP cultures, as assessed by ALP staining (purple), and (b) chondrogenic differentiation of micromass culture assessed by Alcian blue staining. ActA-mAb was used at 1 μg/mL (7 nM) and JAB0505 was used at 10 μg/mL (∼70 nM). (c) Western blot of phosphorylated SMADs1/5/8 (pSMAD1/5/8) in wild-type (WT) and R206H-FAPs (R206H). β-actin was used as a loading control. (d) µCT of the distal hindlimb of Acvr1 tnR206H/+ ;Tie2-Cre mice at day 21 post-injury. At the time of muscle injury, mice were treated with ActA-mAb (10 mg/kg) alone or ActA-mAb with JAB0505 (10 mg/kg). HO is pseudocolored green, and quantification is shown. ActA-mAb, n = 6; JAB0505 + ActA-mAb, n = 6. Bars represent ± SD; p < 0.0001, unpaired t-test. (e) µCT of the distal hindlimb at day 21 post-transplantation of R206H-FAPs into the injured gastrocnemius of SCID hosts. ActA-mAb (10 mg/kg) and JAB0505 (10 mg/kg) were administered at the time of transplantation. HO is pseudocolored green and quantified, with bars representing ± SD. Untreated, n = 10; JAB0505, n = 16; ActA-mAb, n = 6; JAB0505 + ActA-mAb, n = 8.

Article Snippet: A synthetic DNA construct encoding mouse ACVR1 with the R206H mutation (G to A at nucleotide position 617) was assembled from synthetic oligonucleotides and PCR products and cloned into the pCMV6 entry mammalian expression vector (Origene), which also encodes a neomycin resistance marker.

Techniques: Staining, Western Blot, Transplantation Assay

Journal: bioRxiv

Article Title: An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro/adipogenic progenitors in fibrodysplasia ossificans progressiva mice

doi: 10.1101/2021.07.23.451471

Figure Lengend Snippet: ( a ) 3D tomographic bioluminescent source reconstruction following pinch injury of Acvr1 tnR206H/+ ; R26 Luc/+ ;Tie2-Cre FOP mice, with and without administration of JAB0505. Paired images show µCT alone (left panel) and µCT combined with the corresponding 3D bioluminescent reconstruction (right panel). For each experimental group, the same mouse is shown from days 3–21. Bioluminescent reconstruction was not performed at day 21 due to the dampening effect of dense bone on luminescent output. ( b ) Graphical representation of bioluminescent population dynamics of Tie2+ cells from Acvr1 tnR206H/+ ; R26 Luc/+ ;Tie2-Cre mice following pinch injury. Untreated, n = 16; JAB0505, n = 10. Error bars represent ± SEM. (c) Flow cytometry analysis to determine R206H-FAP cell number in injured Acvr1 tnR206H/+ ; R26 NG/+ ;Tie2-Cre which were either untreated (Day 5, n = 4; Day 10, n = 9) or administered JAB0505 at 10 mg/kg (Day 5, n = 4; Day 10, n = 10). p = 0.0032, two-way ANOVA. (d, e) Flow cytometry analysis to determine (d) CD31 cell number and (e) CD45+ cell number 10 days post-injury of Acvr1 tnR206H/+ ; R26 NG/+ ;Tie2-Cre mice that were either untreated (CD31+, n =4; CD45+, n=4) or administered JAB0505 at 10 mg/kg (CD31+, n =4; CD45+, n=4). Error bars represent t ± SD. Significance of (d) p = 0.02 for CD31+ cells and (e) p = 0.05 for CD34+ was determined by unpaired t-test.

Article Snippet: A synthetic DNA construct encoding mouse ACVR1 with the R206H mutation (G to A at nucleotide position 617) was assembled from synthetic oligonucleotides and PCR products and cloned into the pCMV6 entry mammalian expression vector (Origene), which also encodes a neomycin resistance marker.

Techniques: Flow Cytometry

Journal: Scientific Reports

Article Title: LATS kinases and SLUG regulate the transition to advanced stage in aggressive oral cancer cells

doi: 10.1038/s41598-022-16667-5

Figure Lengend Snippet: LATS1 and LATS2 phosphorylate SLUG on T208. ( A ) SAS and SAS-δ cells were co-transfected with siRNAs against LATS1 and LATS2 (siLATS1/2), and then cultured with TGF-β1 for 48 h, followed by WB with the indicated antibodies. ( B ) SAS cells transfected with GL2 (as a negative control) or LATS2 siRNA duplex were treated with cycloheximide (CHX) for the indicated periods, and then subjected to subcellular fractionation. Protein levels of SLUG and LATS2 were determined by WB. Lamin A/C and α-tubulin are nuclear and cytoplasmic fraction markers, respectively. The relative levels of cytoplasmic and nuclear SLUG normalized to the corresponding band intensities of α-tubulin and Lamin A/C, respectively, are shown below top panel. The normalized band intensities of SLUG at 0 h after CHX treatment were defined as 1.0 (lanes 1, 5, 9, and 13). ( C ) WB of parental SAS and SAS-δ, which were cultured in medium containing 10% FBS in the presence (+) or absence (–) of TGF-β1 (48 h), was performed with the indicated antibodies. The relative levels of the indicated proteins normalized to the corresponding band intensity of actin, SLUG, or SMAD2, are shown below panels. ( D ) Immunofluorescence staining with anti-SLUG-pT208 (red) and α-tubulin (green) antibodies. Cells were cultured as in ( C ). DNA was visualized by Hoechst 33258 staining (blue). The cell size were comparable between SAS and SAS-δ in the presence or absence of TGF-β1.

Article Snippet: Recombinant human LATS1 or LATS2 kinase (Carna Biosciences, Hyogo, Japan) was incubated at 30 °C for 30 min with GST-fused mouse Slug-WT, -T183A, -T209A, and all-A (S101A, S139A, S161A, T183A, S196A, T209A, S215A, S248A, and S252A) mutants in LATS1/2-kinase buffer (20 mM PIPES [pH 6.8], 4 mM MnCl 2 , 1 mM DTT, 1 mM NaF, 1 mM Na 3 VO 4 ) containing 20 μM ATP and 10 μCi [γ- 32 P] ATP.

Techniques: Transfection, Cell Culture, Negative Control, Fractionation, Immunofluorescence, Staining